Amidinophenylalanine derivatives as thrombin inhibitors

ABSTRACT

The compounds of the formula (formula 1) and pharmaceutically acceptable salts thereof and a process for preparing the same and pharmaceutical compositions containing the same are described wherein the substituents have the meaning as specified in the description. The compounds are used as thrombon inhibitors.

TECHNICAL FIELD

[0001] The invention belongs to the field of pharmaceutical industry andrelates to novel azaphenylalanine derivatives, procedures for theirpreparation and pharmaceutical compositions containing them. Novelazaphenylalanine derivatives have the enzymatic activity (serineproteases) and anticoagulant effect.

TECHNICAL PROBLEM

[0002] There exists a constant need for new medicinal products withanticoagulant activity that may be administered either orally orparenterally and that are highly selective and of low toxicity.Recently, low molecular weight thrombin inhibitors appear to be ofgrowing importance in this area.

PRIOR ART

[0003] Thrombin as a serine protease is one of key enzymes in theprocesses of blood coagulation and in the development of thrombosis(Edit, J. F.; Allison, P.; Noble, S.; Ashton, J.; Golino, P.; McNard,J.; Buja, L. M.; Willerson, J. T., J. Clin. Invest. 1989, 84, 18.).

[0004] The crystalline structure of serine proteases of thrombin andserine is known (Bode, W.; Turk, D.; Karshikov, A. J., Protein Sci.1992, 1, 426).

[0005] Currently used anticoagulant agents are toxic and of limitedeffectiveness.

[0006] Low molecular weight thrombin inhibitors should have selectiveefficacy and can be used in oral administration. There are reversibleand irreversible thrombin antagonists (Kimball, S. D., Curr. Pharm.Design 1995, 1, 441, Das, J.; Kimball, S. D., Bioorg. Med. Chem. 1995,3, 999, Kimball, S. D., Blood Coagulation and Fibrinolysis 1995, 6, 511,Breznik, M.; Peèar, S., Farm. Vestn. 1997, 48, 545, Sanderson, P. E. J.;Naylor-Olsen, A. M.; Current Med. Chem. 1998, 5, 289, Rewinkel, J. B.M.; Adang, A. E. P., Curr. Pharm. Design 1999,5,1043, Wiley, M. R.;Fisher, M. J.; Exp. Opin. Ther. Patents 1997, 7, 1265, Menear, K.;Current Med. Chem. 1998, 5, 457.

[0007] The majority of reversible thrombin antagonists derive frompeptidomimetically modified structure D-Phe-Pro-Arg. The aim of themodification is to provide chemical stability, selectivity andeffectiveness.

[0008] The active substance used in Japan and the USA is argatroban(Kikumoto, R.; Tamao, Y.; Tezuka, T.; Tonomura, S.; Hara, H., Ninomiya,K.; Hijikata, A.; Okamoto, S., Biochemistry 1984, 23, 85).

[0009] Amidinophenylalanine derivatives and aminopyridylalaninederivatives have selective antithrombotic activity (Danilewicz, J., etal. WO 95/13274 (1995)).

[0010] By replacing amidino-pyperidine P1 group with a weakly basic2-amino-6-metylpyridine structure, a selective and effective thrombininhibitor that can be used in oral administration has been obtained(Sanderson, P. E. J.; et al., Bioorg. Med. Chem. 1998, 8, 817).

[0011] SI 20025 describes thrombin inhibitors which are the derivativesof azaphenylalanine.

[0012] Accordingly, the object of the present invention is to overcomethe problems encountered in the prior art.

DESCRIPTION OF THE SOLUTION OF TECHNICAL PROBLEM INCLUDING EXAMPLES

[0013] The above problems are solved e.g. by a compound as defined inclaim 1 and a process as defined in claim 6 as well as pharmaceuticalcompositions as defined in claim 8.

[0014] The invention relates particularly to novel azaphenylalaninederivatives and analogs thereof of the general formula (I)

[0015] wherein

[0016] R¹ represents a residue of the formula

[0017]  wherein R⁴=H, alkyl (C₁-C₃), OH, O-alkyl (C₁-C₃), NH₂;

[0018] R² represents a residue of the formula

[0019]  wherein

[0020] R⁵H, alkyl (C1-C3), CF₃, COOR⁹,

[0021] R⁶=H, alkyl (C₁-C₃), CF₃, COOR⁹,

[0022] R⁷=alkyl (C1-C3), cycloalkyl (C3-C0),

[0023] R⁸=alkyl (C₁-C₃), cycloalkyl (C3-C6),

[0024] R⁹=H, alkyl (C₁-C₃),

[0025] R¹⁰=H, alkyl (C₁-C₃), benzyl

[0026] R¹¹=H, SO₂Me, CO₂Me;

[0027] R³ represents a residue of the formula

[0028] wherein R¹²=H, alkyl (C₁-C₃), acetyl

[0029] and pharmaceutically acceptable salts thereof.

[0030] In comparison to the compounds, described in SI 20025, thecompounds of the present invention are of greater activity. Ki thrombin(μM) of the compound a, according to the patent SI 20025, is 0.032, Kithrombin (μM) of the compound b, according to the present invention, is0.005.

[0031] The invention also particularly relates to a process for thepreparation of azaphenylalanine derivatives and analogs of the generalformula (I).

[0032] They may be suitably prepared as follows:

[0033] a) 3-cyanobenzaldehyde of formula (II)

[0034] is converted with BOC-carbazate of the formula (III)

[0035] to the compound of the formula (IV),

[0036] which is subjected to reduction by catalytic hydrogenation orwith NaCNBH₃ and is converted to the compound (V),

[0037] which reacts with triphosgene and amine of the formula (VI),

[0038] wherein R⁵ and R⁶ have the same meanings as in the formula (I),

[0039] or with triphosgene and amine of the formula (VII or VIII),

[0040] or with triphosgene and amine of the formula (IX),

[0041] wherein R⁷ and R⁸ have the same meanings as in the formula (I),

[0042] or with triphosgene and amine of the formula (X),

[0043] wherein R¹⁰ has the same meaning as in the formula (I),

[0044] in “one pot” reaction to the compound (XI)

[0045] wherein R² has the same meaning as in the formula (I).

[0046] The protecting BOC group in the compound (XI) is removed with HCl(g) in HOAc at room temperature to obtain the compound (XII),

[0047] wherein R² has the same meanings as in the formula (I)

[0048] which reacts with aromatic sulfonylchloride up to the compound(XIII),

[0049] wherein R² and R³ have the same meanings as in the formula (I).

[0050] The compound (XIII) is converted to the compound of the formula(I)

[0051] in a usual manner. This can be achieved e.g. by convertingcompound (XIII) by addition of a hydroxylamine and an anhydrous alcoholsuch as e.g.—ethanol or a mineral acid such as e.g. HCl(g) and e.g.ammonium acetate, thus obtaining compound (I).

[0052] When the compound (XIII) reacts with hydroxylamine in anhydrousethanol, R⁴ denotes OH group, after the reaction of the compound (XIII)with HCl (g) and ammonium acetate R⁴ denotes hydrogen.

[0053] The starting compounds are prepared, unless otherwise directed,according to the procedures described in the literature; e.g., thecompound of the formula IV as described by A. Fäissler, et. al., J. Med.Chem. 1996, 39, 3203-3215.

[0054] The invention further relates to the use of compounds of theformula I as therapeutically active compounds. The novel compounds arepreferably thrombin inhibitors. They inhibit thrombin and formation offibrin. They are useful in the treatment or prevention of a variety ofthrombosis forms: (i) venous thromboembolism due to formation of athrombus within a vein (venous thrombosis) associated with acquired(prolonged bedrest, surgery, injury, malignancy, pregnancy andpostpartum states or inherited (deficiency of natural coagulationinhibitors) risk factors, obstruction or occlusion of a lung artery by adetached thrombus (pulmonary embolism), (ii) cardiogenic thromboembolismdue to formation of a thombus in the heart associated with cardiacarrythmia, heart valve defect, prosthetic heart valves or heart disease,embolism of peripheral arteries caused by a detached thrombus, mostcommonly in the brain (ischemic stroke), (iii) arterial thrombosis dueto underlying atherosclerotic processes in the arteries which obstructsor occludes an artery and causes myocardial ischemia (angina pectoris,acute coronary syndrome) or heart muscle death cell (myocardialinfarction), obstructs or occludes a peripheral artery (ischemicperipheral artery disease) and obstructs or occludes the artery afterthe procedure on the blood vessel (reocclusion or restenosis aftertransluminal coronary angioplasty, reocclusion or restenosis afterpercutaneous transluminal angioplasty of peripheral arteries) and (iv)in the number of states (e.g., in complications in pregnancy, inmetastazing malignant diseases, after extensive injuries, in bacterialsepsis) when thrombogenic activation causes widespread formation ofthrombi within the vascular system (disseminated intravascularcoagulation).

[0055] The compounds of the present invention may be also used as anadjunct therapy in conjunction with thrombolytic therapy in recentmyocardial infarction, in combination with aspirin in patients withunstable angina pectoris designed to undergo percutaneous transluminalangioplasty and in the treatment of patients with thrombosis and withheparin-induced thrombocytopenia.

[0056] The compounds of the present invention may further be used forthe prevention of coagulation of blood which is in contact withnonbiological surfaces (vascular prosthesis, vascular stents, prostheticheart valves, extracorporeal circulation systems, hemodialysis) and invitro to prevent coagulation in biological samples for testing orstorage.

[0057] The present invention also relates particularly to pharmaceuticalcompositions comprising the compounds of the formula I. They can beformulated as injectable or oral formulations. The active drug componentmay be combined with suitable standard additives with respect to theintended form of administration. The pharmaceutical compositions may beprepared according to the standard procedures. The preparation may beformulated in such a manner as to permit controlled and sustainedrelease of the active ingredient. Dosage, frequency and mode ofadministration depend on a variety of factors, they also depend onindividual active ingredient and its pharmacokinetic parameters and onthe patient's therapeutic needs.

[0058] In Vitro Assays for Determining Proteinase Inhibition

[0059] Coagulation Assays

[0060] The Anticoagulant effect of thrombin inhibitors was measured bythrombin time (TT), activated partial thromboplastin time (aPTT), andprothrombin time (PT) assays. Inhibitors were added to normal pooledhuman plasma over a range of concentrations and clotting was recorded inan automated coagulometer (Fibrintimer, Dade/Behring). The concentrationof inhibitor that doubled the clotting time was determined for eachassay.

[0061] 1. Thrombin Time (TT)

[0062] a) Principle of Method

[0063] In a plasma sample thrombin converts fibrinogen into fibrin andthe clot is formed. The time for clot formation was measured. Thrombintime is prolonged due to disorders in fibrin polymerisation or due tothe presence of thrombin inhibitors.

[0064] b) Reagents

[0065] Thrombin (Test Thrombin Reagent, 1.5 IU/mL, Dade/Behring):lyophilized bovine thrombin was dissolved in 5 mL of HEPES (25 mmol/L,pH 7.4). The reagent was warmed to 37° C. prior to the assay.

[0066] Normal pooled plasma: venous blood from 11 apparently healthyvolunteers was collected (1 part 0.11 mol/L sodium citrate solution with9 parts of blood) and centrifuged immediately at 2000×g for 30 minutesat 4° C. Plasma was removed, pooled and stored in aliquots at −70° C.until use.

[0067] Inhibitors: were dissolved in DMSO (10 mM stock solution) anddiluted with distilled water to working solutions (the highestconcentration 100 μM).

[0068] c) Method

[0069] Inhibitors (10 μL working solution, concentrations from 2.5 μM to100 μM) and pooled plasma (90 μL) were incubated at 37° C. for 5 minutesand then thrombin (200 μL) was added. Clot formation was measured in acoagulometer in duplicate.

[0070] 2. Activated Partial Thromboplastin Time (aPTT)

[0071] a) Principle of Method

[0072] Incubation of plasma with the optimal quantity of phospholipidsand a surface activator leads to activation of factors of the intrinsiccoagulation pathway. The addition of calcium ions triggers thecoagulation process. The time of fibrin clot formation wass measured.aPTT was used as a screening test for coagulation disorders of theintrinsic coagulation pathway. It is prolonged due to intrinsiccoagulation factors deficit or due to the presence of inhibitors.

[0073] b) Reagents

[0074] Pathromtin SL (Dade/Behring): silicon dioxide particles,vegetable phospholipids, sodium chloride (2.4 g/L), Hepes (14.3 g/L, pH7.6), sodium azide (<1 g/L). The reagent was used at room temperature(15-25° C.).

[0075] Calcium chloride solution: 0.025 mol/L, warmed to 37° C.

[0076] Normal pooled plasma: venous blood from 11 apparently healthyvolunteers was collected (1 part 0.11 mol/L sodium citrate solution with9 parts of blood) and centrifuged immediately at 2000×g for 30 minutesat 4° C. Plasma was removed, pooled and stored in aliquots at −70° C.until use.

[0077] Inhibitors: were dissolved in DMSO (10 mM stock solution) anddiluted with distilled water to working solutions (the highestconcentration 100 μM).

[0078] c) Method

[0079] Inhibitor (10 μL working solution, concentrations from 5 μM to100 μM) and pooled plasma (90 μL) were incubated at 37° C. for 5minutes. Pathromtin (100 μL) was added and the sample was incubated foranother 2 minutes at 37° C. The addition of calcium chloride (100 μL)triggered the coagulation process and clot formation was detected with acoagulometer in duplicate.

[0080] 3. Prothrombin Time (PT)

[0081] a) Principle of Method

[0082] An optimal amount of thromboplastin and calcium are added toplasma sample and the time of fibrin clot formation wass measured. PT isa rapid, sensitive screening test for coagulation disorders of theextrinsic pathway. It is well suited for the induction and monitoring oforal anticoagulant therapy, for diagnosing genetic or acquireddeficiencies in coagulation factors and checking the synthesisperformance of the liver in hepatic diseases. PT is prolonged due toextrinsic coagulation factors deficit or due to the presence ofinhibitors.

[0083] b) Reagents

[0084] Thromboplastin (Thromborel S, Dade/Behring): was dissolved in 4mL of distilled water. Reagent was at 37° C. prior to use at least 30minutes.

[0085] Normal pooled plasma: venous blood from 11 apparently healthyvolunteers was collected (1 part 0.11 mol/L sodium citrate solution with9 parts of blood) and centrifuged immediately at 2000×g for 30 minutesat 4° C. Plasma was removed, pooled and stored in aliquots at −70° C.until use.

[0086] Inhibitors: were dissolved in DMSO (10 mM stock solution) anddiluted with distilled water to working solutions (the highestconcentration 100 μM).

[0087] c) Method

[0088] Inhibitor (10 μL working solution, concentrations from 5 μM to100 μM) and pooled plasma (90 μL) were incubated at 37° C. for 5 minutesand then Thromborel S (200 μL) was added. Clot formation was measuredwith a coagulometer in duplicate.

[0089] Enzyme Assay

[0090] The enzyme inhibitory effect of the compounds can be identifiedby determination of inhibition constant Ki. It denotes the degree ofdissociation of the enzyme-inhibitor complex. Low dissociation constantmeans high potency of inhibitor. Ki can be determined during reaction ofthe enzyme with a specific chromogenic substrate which under hydrolysisby the enzyme develops color. Reaction in time is recorded byspectrophotometry and Ki is calculated from kinetic parameters (Vmax,Km, reaction rate).

[0091] 1. Determination of Thrombin Ki

[0092] a) Principle of Method

[0093] The ability of a compound to act as an inhibitor of humanthrombin catalytic activity was assessed by determination of Ki.

[0094] b) Reagents

[0095] Buffer HBSA, pH 7.5 (10 mM Hepes, 150 mM NaCl, 0.1% w/v bovineserum albumin)

[0096] Substrate (S-2238: H-D-Phe-Pip-Arg-pNA HCl, 25 mg; Chromogenix):dissolved in distilled water to 1 mM concentration. (Km is 2.6 μM)

[0097] Human thrombin (308 NIH; Sigma): dissolved in saline to give astock solution of 20 NIH/mL.

[0098] Inhibitors: were dissolved in DMSO (10 mM stock solution) anddiluted with distilled water to working solutions (the highest finalconcentration of DMSO was 3%).

[0099] c) Method

[0100] A mixture of 50 μL of buffer, 50 μL of inhibitor in water (finalconcentrations from 10 to 100 μM) and 50 μL of thrombin (0.5 NIH U/mLf.c.) was incubated for 15 minutes at room temperature. The reaction wasstarted with 50 μL of S-2238 (20 μM or 40 μM f.c.) and the absorbance ofeach sample at 405 nm (at 25° C.) was measured in triplicate every 10seconds for a period of 15 minutes using a microtiter plate reader(Tecan Sunrise).

[0101] Thrombin activity was determined from the change in absorbance inthe linear part of the velocity graph. Ki was calculated according toCheng and Prussof (Biochem Pharmacol, 1973) where Ki is IC₅₀/(1+S/Km).The Km for the substrate was determined under the test conditions withat least 6 substrate concentrations varying around Km and calculatedwith the non-linear regression programme Curve expert.

[0102] 2. Determination of Trypsin Ki

[0103] a) Principle

[0104] The ability of a compound to act as an inhibitor of trypsincatalytic activity was assessed by determination of Ki.

[0105] b) Reagents

[0106] Buffer: HBSA, pH 7.5 (10 mM Hepes, 150 mM NaCl, 0.1% w/v bovineserum albumin)

[0107] Substrate (S-2222: N-benzoyl-lle-Glu-Gly-Arg-pNA HCl, 25 mg;Chromogenix): dissolved in distilled water to 2 mM concentration. (Km is21 μM)

[0108] Trypsin (6000 E/mg prot.; Sigma): dissolved in distilled water togive a stock solution of 300 E/mL.

[0109] Inhibitors: were dissolved in DMSO (10 mM stock solution) anddiluted with distilled water to working solution (the highest finalconcentration of DMSO was 10%).

[0110] c) Method

[0111] 50 μL of buffer, 50 μL of test compound in water (finalconcentrations from 50 to 300 μM) and 50 μL of trypsin (3 mE/mL f.c.)were incubated for 15 minutes at room temperature. The reaction wasstarted with 50 μL of S-2222 (50 μM or 100 μM f.c.) and the absorbanceof each sample at 405 nm (at 25° C.) was measured in triplicates every10 seconds for a period of 15 minutes using a microtiter plate reader(Tecan Sunrise).

[0112] Trypsin activity was determined from the change in absorbance inthe linear part of velocity graph. Ki was calculated according to Chengand Prussof (Biochem Pharmacol, 1973) where Ki is IC₅₀/(1+S/Km). The Kmfor the substrate was determined under test conditions with at least 6substrate concentrations varying around Km and calculated with thenon-linear regression program Curve expert.

[0113] 3. Selectivity to Thrombin

[0114] By the use of other serine protease like trypsin with theappropriate chromogenic substrate, selectivity of inhibitors withrespect to thrombin was determined. Selectivity of an inhibitor isexpressed as a ratio of Ki for trypsin to Ki for thrombin.

[0115] In vivo Assays

[0116] 1. Stasis-Induced Thrombosis

[0117] Principle of Method

[0118] Using a combination of thrombogenic challenge (0.045 ng/kg oftissue factor) and stasis, compounds were tested for their abillity toaffect thrombus formation in a venous thrombosis model in the rat.

[0119] Method

[0120] Thrombus formation by a combination of stasis andhypercoagulability was induced as described by Vogel et al. (Thromb.Res., 1989, 54, 399410). Male sprague-Dawley rats (250-300 g) wereanaesthetized with sodium pentobarbitone (30 mg/kg, i.p.). The abdomenof the animals was surgically opened and after careful dissection, thevena cava was exposed and dissected free from surrounding tissue.Saline, or the various compounds were administered i.v. 5 min beforethrombosis induction. Two loose sutures were prepared 0.7 cm apart onthe inferior vena cava and all collateral veins were ligated. Humantissue factor (0.045 ng/kg, i.v.) was injected into the dorsal vein ofthe penis. Ten seconds after the end of the injection, stasis wereestablished by tightening the two, firstly the proximal and then thedistal. The abdominal cavity was provisionally closed and stasis wasmaintained for 20 minutes. The cavity was then reopened, the ligatedsegment was opened longitudinally and the thrombus formed was removed,rinsed, blotted on filter paper, dried overnight at 60° C. and weighed.Under these experimental conditions, control thrombus weight was 5.5±0.1mg (n=15).

[0121] 2. Bleeding Time

[0122] Principle of Method

[0123] The hemorrhagic risk associated to a treatment with selectedcompounds were determined using an experimental model of bleeding:transection of the tail of rats.

[0124] Method

[0125] Bleeding time was determined by transection of the tail, 2 mmfrom the tip, of phenobarbital-anaestetised rats (30 mg/kg i.p.). Thecompounds were injected i.v. at the indicated doses 5 min before tailtransection. Blood was carefully blotted every 15 s on a filter paper.Haemostasis was considered to be achieved when no more blood stain wasobserved over 1 min.

EXAMPLES

[0126] The invention is further described by means of the followingexamples, but not in any limitative case:

Example 1

[0127] tert-Butyl2-(3-cyanobenzyl)-2-[(4-methyl-1-piperidinyl)carbonyl]-1-hydrazinecarboxylate

[0128] Triphosgene (2.296 g) was dissolved in 15 ml of anhydrousdichloromethane and cooled to −5° C. While stirring under argonatmosphere, a solution ofN-1-(tert-butoxycarbonyl)-N-2-[(3-cyanophenyl)methyl]hydrazine (3.833 g)and N, N-diisopropylethylamine (4.040 ml) in 25 ml of dichloromethanewas added dropwise. After addition, the reaction mixture was stirred foranother 5 minutes, and a mixture of 4-methyl piperidine (1.836 ml) andN,N-diisopropylethylamine (4.040 ml) in 25 ml dichloromethane was thenadded at once and stirred at room temperature for another 20 minutes.The solvent was evaporated in vacuo and the residue was dissolved in 50ml of ethyl acetate, washed with 25 ml of 5% citric acid, with 25 ml of5% solution of NaHCO₃ and with 25 ml of brine. The organic phase wasdried over MgSO₄, filtered and the filtrate evaporated under reducedpressure.

[0129] The resulting mixture was suspended in 20 ml of diethylether and5 ml of hexane was added. The precipitate was filtered by suction.

[0130] Yield: 1.756 g (30.4%)

[0131] Melting range: 147-150° C.

[0132] IR (KBr, cm⁻¹): 3275.0, 2922.7, 2229.3, 1722.3, 1632.3, 1497.3,1445.5, 1367.6, 1252.9, 1157.0, 979.2, 805.1, 736.2, 573.7

[0133] NMR (DMSO-d₆)): δ (ppm): 0.86 (d, J=6,4 Hz, 3H, CH₃), 0.90-1.05(m, 2H, Pip-H^(3,5)), 1.35 (s, 9H, Boc), 1.45-1.60 (m, 4H,Pip-H^(3′,5′)), 2.60-2.75 (m, 2H, Pip-H^(2,6)), 3.70-3.85(m, 2H,Pip-H^(2′,6′)), 4.52 (br s, 2H, CH₂), 7.58-7.66 (m, 1H, Bn-H⁵),7.79-7.85 (m, 1H, Bn-H⁶), 7.93-8.05 (m, 2H, Bn-H^(2,4)), 11.05 (s, 1H,NH).

[0134] Molecular weight of C₂₀H₂₇ N₄ O₃: calculated: 372; found: 373(MH⁺)

Example 2

[0135] 2-(3-Cyanobenzyl)-2-[(4-methyl-1-piperidinyl)carbonyl]hydraziniumChloride

[0136] terc-butyl2-(3-cyanobenzyl)-2-[(4-methyl-1-pyperidinyl)carbonyl]hydrazinecarboxylate (1.173 mg) was dissolved in 30 ml of glacial acetic acid andHCl gas was bubbled through the solution for 20 minutes. The progress ofthe reaction was monitored by TLC. Glacial acetic acid was evaporated ona rotavapor, and diethylether was poured over the oily product. Theliquid phase was removed, the resulting precipitate is dried at 50° C.and desiccated over NaOH for 24 hours.

[0137] Yield: 0.875 g (%) of the mixture

[0138] Melting range: 170-173° C.

[0139] IR (KBr, cm⁻¹): 3417.8, 2934.6, 2664.1, 2227.3, 1711.6, 1641.3,1433.7, 1402.3, 1249.6, 1132.0, 1017.8

[0140] NMR (DMSO-d₆): δ (ppm): 0.92 (d, J=6,4 Hz, 3H, CH₃), 0.99-1.16(m, 2H, Pip-H^(3,5)), 1.52-1.71 (m, 4H, Pip-H^(3′,5′)), 2.90-3.05 (m,2H, Pip-H^(2,6)), 3.80-3.95(m, 2H, Pip-H^(2′,6′)), 4.62 (s, 2H, CH₂),7.59-7.69 (m, 1H, Bn-H^(4,5)), 7.79-7.88 (m, 1H, Bn-H^(2,6)).

[0141] Molecular weight of C₁₄H₁₉ClN₄O: calculated: 294.78; found:295(MH⁺)

Example 3

[0142]N′-(3-Cyanobenzyl)-N′-[(4-methyl-1-piperidinyl)carbonyl]-2-naphthalenesulfonohydrazide

[0143] 2-(3-cyanobenzyl)-2-[(4-methyl-1-pyperidinyl)carbonyl]hydrazine(0.870 g) was dissolved in 50 ml of dichloromethane, cooled to −5° C.and while stirring, 1.190 ml of triethylamine and 0.640 g ofnaphthalen-2-sulfonylchloride were added. The reaction mixture wasstirred at −5° C. for 30 minutes and then at room temperature foranother 24 hours. The solvent was evaporated under reduced pressure and100 ml of ethyl acetate was added to the residue. In the separatoryfunnel it was washed twice with 50 ml of water, with 50 ml of 10% ofcitric acid and with 50 ml of brine. The organic phase was dried overNa₂SO₄, filtered and the filtrate was evaporated in vacuo. The resultingproduct was suspended in diethylether and the precipitate was filteredby suction. The precipitate was then recrystalized from ethanol and thecrystals were filtered by suction.

[0144] Yield: 0.464 g (35.6%)

[0145] Melting range: 92-95° C.

[0146] IR (KBr, cm⁻¹): 3191.7, 2931.6, 2221.0, 1680.5, 1433.5, 1333.1,1168.8, 857.9, 749.4, 692.2

[0147]¹H NMR (DMSO-d₆)): δ (ppm): 0.10-0.35 (m, 2H, Pip-H^(3′,3′,5,5′)),0.47 (d, 3H, J=6.0 Hz, CH₃), 1.22-1.32 (m, 3H, Pip-H^(3,3′,4,4′,5,5′)),2.30-2.45 (m, 1H, Pip-H^(2,2′)), 2.65-2.80 (m, 1H, Pip-H^(6,6′)),3.45-3.65 (m, 2H, Pip-H^(2,2′,6,6′)), 4,38 (br d, 2H, CH₂), 7.47-7.55(m, 2H, Bn-H^(5,6)), 7.58-7.63 (m, 1H, Bn-H²), 7.63-7.80 (m, 4H, Bn-H⁴in Nf-H_(3,6,7)), 8.02-8.18 (m, 3H, Nf-H^(4,5,8)), 8.43 (s, IH, Nf-H¹),9.60 (s, 1H, NH). Two sets of signals

[0148] Molecular weight of C₂₅H₂₆N₄O₃S: calculated: 462.6; found: 463(MH⁺)

Example 4

[0149]N′-Hydroxy-3-{[1-[(4-methyl-1-piperidinyl)carbonyl]-2-(2-naphthylsulfonyl)hydrazino]methyl}benzenecarboximidamide

[0150]N′-(3-cyanobenzyl)-N′-[(4-metyl-1-pyperidinyl)carbonyl]-2-naphthalenesulfonohydrazide (0.223 g) was suspended in 20 ml of anhydrous ethanolunder argon atmosphere and 0.032 g of hydroxylamine was added. Thereaction mixture was heated at reflux temperature for 24 hours andevaporated under reduced pressure. The resulting product was purified bycolumn chromatography (eluent:chloroform/methanol=9/1) to yield whitecrystals.

[0151] Yield: 0.215 g (38%)

[0152] Melting range: 169-173° C.

[0153] IR (KBr, cm⁻¹): 3365.8, 3194.6, 2943.8, 2864.7, 1652.6, 1447.5,1332.2, 1169.3, 1076.1, 756.2, 700.3, 553.2

[0154]¹H-NMR (DMSO-d₆): δ (ppm): 0.01-0.36 (m, 2H, Pip-H^(3,5)), 0.44(d, 3H, J=6.4 Hz, CH₃), 1.15-1.27 (m, 3H, Pip-H^(3,4,5)), 2.27-2.42 (m,1H, Pip-H²), 2.56-2.75 (m, 1H, Pip-H⁶), 3.45-3.63 (m, 2H, Pip-H ^(2,6)),4.35 (br d, 2H, CH₂), 7.14 (d, 1H, J=7.9, Bn-H⁴), 7.25-7.40 (m, 1H,Bn-H⁵), 7.48-7.59 (m, 2H, Bn-H^(2,6)), 7.63-7.81 (m, 3H, Nf-H^(3,6,7)),8.02-8.19 (m, 3H, Nf-H^(4,5,8)), 8.46 (s, 1H, Nf-H¹), 9.40 (s, 1H, NH),9.78 (s, 1H, OH).

[0155] Molecular weight of C₂₅H₃₀N₅O₄S: calculated: 495.6; found: 496(MH⁺)

Example 5

[0156]Imino(3{[1-[(4-methyl-1-piperidinyl)carbonyl]-2-(2-naphthylsulfonyl)hydrazino]methyl}phenyl)methanaminiumChloride

[0157]N′-(3-cyanobenzyl)-N′-[(4-methyl-1-pyperidinyl)carbonyl]-2-naphthalenesulfonohydrazide (0.200 g) was suspended in 10 ml of ethanol, cooled inan ice bath and HCl was bubbled into the suspension for 30 minutes. Themixture was stirred at room temperature for 4 hours and then the solventwas evaporated under reduced pressure. The solid residue was washed withisopropyl ether and filtered by suction. The resulting precipitate wasdissolved in 10 ml ethanol and ammonium acetate (0.092 g) was added. Theresulting mixture was stirred at room temperature for 20 hours and thenHCl was passed through the solution for 30 minutes. After 24 hours thewhite precipitate was formed which was filtered by suction.

[0158] Yield: 50.4 mg (52%)

[0159] Melting range: 148-152° C.

[0160] IR (KBr, cm⁻¹): 3396.1, 3101.7, 2945.8, 1669.1, 1435.8, 1335.9,1165.9, 1072.4, 749.4, 668.4

[0161]¹H-NMR (DMSO-d₆): δ (ppm): 0.01-0.40 (m, 2H, Pip-H^(3,5)), 0.47(d, 3H, J=6.4 Hz, CH₃), 1.15-1.29 (m, 3H, Pip-H^(3,4,5)), 2.27-2.42 (m,1H, Pip-H ²), 2.56-2.75 (m, 1H, Pip-H⁶), 3.45-3.60 (m, 2H, Pip-H^(2,6)),4.41 (br d, 2H, CH₂), 7.14 (d, 1H, J=7.9, Bn-H⁴), 7.25-7.40 (m, 1H,Bn-H⁵), 7.48-7.59 (m, 2H, Bn-H^(2,6)), 7.63-7.81 (m, 3H, Nf-H^(3,6,7)),8.02-8.19 (m, 3H, Nf-H^(4,5,8)), 8.46 (s, 1H, Nf-H¹), 9.40 (s, 1H, NH).

[0162] Molecular weight of C₂₄H₂₇N₅O₃S (free base): calculated: 465.57;found: 466 (MH⁺)

Example 6

[0163] tert-Butyl 2-(3-cyanobenzylidene)-1-hydrazinecarboxylate

[0164] 3-Cyanobenzaldehyde (40.0 mmol) suspended in EtOH (100 mL) wasadded to a stirred solution of tert-butylcarbazate (40.0 mmol) in EtOH.The mixture was heated at reflux temperature and after 4 h the EtOH waspartially evaporated in vacuo. Water (100 mL) was added and theprecipitated product was collected by filtration and washed withdiethylether to give tert-butyl2-(3-cyanobenzylidene)-1-hydrazinecarboxylate as a white solid.

[0165] Yield: 77%.

[0166] MP: 115-158° C.

[0167] IR (KBr): 3297, 2977, 2232, 1703, 1530, 1477, 1376, 1248, 1160,1061, 941, 865 cm⁻¹.

[0168] MS (FAB): MH⁺=246.

[0169]¹H NMR (300 MHz CDCl₃): 1.55 (s, 9H, Boc-H), 7.55 (m, 2H, Bn-H),7.93 (m, 3H, Bn-H in CH), 8.24 (s, 1H, NH).

[0170] Elemental analysis: calculated for C₁₃H₁₅N₃O₂: C, 63.66; H, 6.16;N, 17.13, found C, 63.38; H, 6.18; N, 17.09.

Example 7

[0171] tert-Butyl 2-(3-cyanobenzyl)-1-hydrazinecarboxylate

[0172] tert-Butyl 2-(3-cyanobenzylidene)-1-hydrazinecarboxylate (60.0mmol) was dissolved in MeOH (250 mL) and 10% Pd/C (10 w/w %) was added.The mixture was hydrogenated for 6 h. The catalyst was filtered off andthe filtrate was evaporated in vacuo to yield oily tert-butyl2-(3-cyanobenzyl)-1-hydrazinecarboxylate which crystallized overnight.

[0173] Yield: 95%.

[0174] MP: 75-80° C.

[0175] IR (KBr): 3368, 2980, 2226, 1679, 1482, 1366, 1286, 1166, 793cm⁻¹

[0176] MS (FAB): MH⁺=248.

[0177]¹H NMR (300 MHz CDCl₃): 1.47 (s, 9H, Boc-H), 4.04 (br s, 2H, CH₂),4.28 (br s, 1H, NH), 6.09 (br s, 1H, NH), 7.12-7.41(m, 4H, Bn-H).

[0178] Elemental analysis: calculated for C₁₃H₁₇N₃O₂·¼ H₂O: C, 62.01; H,7.00; N. 16.69; found C, 61.90; H, 7.22; N, 16.69.

Example 8

[0179] tert-Butyl2-(3-cyanobenzyl)-2-[(2-methyl-1-piperidinyl)carbonyl]-1-hydrazinecarboxylate

[0180] The product was prepared from tert-butyl2-(3-cyanobenzyl)-1-hydrazinecarboxylate and 2-methylpiperidine usingthe procedure described in EXAMPLE 1.

[0181] Yield: 42%.

[0182] MP: oil

[0183] IR (KBr): 3271, 2941, 2230, 1725, 1644, 1428 cm⁻¹.

[0184] MS (FAB): MH⁺=373.

[0185]¹H NMR (300 MHz, CDCl₃): 1.22 (d, J=6,8 Hz, 3H, CH₃), 1.45 (s, 9H,Boc), 1.45-1.79 (m, 5H, Pip-H), 2.97-3.10 (m, 1H, Pip-H), 3.44-3.54 (m,1H, Pip-H), 3.63-3.76 (m, 1H, Pip-H), 4.18-4.32 (m, 1H, Pip-H), 4.48 (s,2H, CH₂), 6.30 (s, 1H, NH), 7.46 (t, J=7.9 Hz, 1H, Bn-H), 7.56-7.67 (m,3H, Bn-H).

Example 9

[0186] 2-(3-Cyanobenzyl)-2-[(2-methyl-1-piperidinyl)carbonyl]hydraziniumChloride

[0187] The product was prepared from tert-butyl2-(3-cyanobenzyl)-2-[(2-methyl-1-piperidinyl)carbonyl)-1-hydrazinecarboxylateusing the procedure described in EXAMPLE 2

[0188] Yield: 91%.

[0189] MP: 135-137° C.

[0190] IR (KBr): 3429, 2949, 2229, 2708, 2230, 1694, 1526, 1420 cm⁻¹.

[0191] MS (FAB): MH⁺=273.

[0192]¹H NMR (300 MHz, CDCl₃): 1.23 (d, J=6,8 Hz, 3H, CH₃), 1.40-1.51(m, 1H, Pip-H) 1.52-1.79 (m, 5H, Pip-H), 2.98-3.10 (m 1H, Pip-H),3.60-3.79 (m, 1H, Pip-H), 4.25-4.38 8 (m, 1H, Pip-H), 4.64 (s, 2H, CH₂),7.53 (t, J=7.9 Hz, 1H, Bn-H), 7.61-7.76 (m, 3H, Bn-H), 9.60 (br s, 2H,NH₂).

Example 10

[0193] tert-Butyl2-(1-azepanylcarbonyl)-2-(3-cyanobenzyl)-1-hydrazinecarboxylate

[0194] The product was prepared from tert-butyl2-(3-cyanobenzyl)-1-hydrazinecarboxylate and azepane using the proceduredescribed in EXAMPLE 1.

[0195] Yield: 63%.

[0196] MP: oil.

[0197] IR (KBr): 3287, 2925, 2223, 1726, 1634 cm⁻¹.

[0198] MS (FAB): MH⁺=373.

[0199]¹H NMR (300 MHz, CDCl₃): 1.45 (s, 9H, Boc), 1.53-1.62 (m, 4H,Pip-H^(4,5)), 1.69-1.80 (m, 4H, Pip-H^(3,6)), 3.39-3.46 (m, 4H,Pip-H^(2,7)), 4.50 (s, 2H, CH₂), 6.25 (br s, 1H, NH), 7.45 (t, J=7.9 Hz,1H, Bn-H), 7.56-7.69 (m, 3H, Bn-H).

Example 11

[0200] 2-(1-Azepanylcarbonyl)-2-(3-cyanobenzyl)hydrazinium Chloride

[0201] The product was prepared from tert-butyl2-(1-azepanylcarbonyl)-2-(3-cyanobenzyl)-1-hydrazinecarboxylate usingthe procedure described in EXAMPLE 2.

[0202] Yield: 66%.

[0203] MP: oil.

[0204] IR (KBr): 2926, 2702, 2230 1703, 1521 cm⁻¹.

[0205] MS (FAB): MH⁺=273.

[0206]¹H NMR (300 MHz, CDCl₃): 1.53-1.64 (m, 4H, Pip-H^(4,5)), 1.65-1.81(m, 4H, Pip-H^(3,6)), 3.38-3.46 (m, 4H, Pip-H^(2,7)), 4.70 (s, 2H, CH₂),7.48-7.64 (m, 2H, Bn-H), 7.69-7.80 (m, 2H, Bn-H), 9.60 (br s, 2H, NH₂).

Example 12

[0207]N′-(3-cyanobenzyl)-N′-[(2-methyl-1-piperidinyl)carbonyl]-2-naphthalenesulfonohydrazide

[0208] The product was prepared from2-(3-cyanobenzyl)-2-[(2-methyl-1-piperidinyl)carbonyl]hydraziniumchloride and naphthalene-2-sulfonyl chloride using the proceduredescribed in EXAMPLE 3.

[0209] Yield: 14%.

[0210] MP: 116-119° C.

[0211] IR (KBr): 3231, 2943, 2227, 1653 cm⁻¹.

[0212] MS (FAB): MH⁺=273.

[0213]¹H NMR (300 MHz CDCl₃): 0.35-0.55 (m, 2H, Pip-H), 0.75-0.90 (m,2H, Pip-H), 1.03-1.45 (m, 6H, Pip-H in CH₃), 2.54-2.70 in 2.75-2.95 (m,1H, Pip-H) 3.50-3.75 in 3.75-4.00 (m, 1H, Pip-H), 4.31 (br d, 2H, CH₂),7.35-7.70 (m, 7H, Bn-H in Nph-H), 7.75-7.85(m, 1H, Nph-H), 7.86-8.00 (m,3H, Nph-H), 8.42 (s, 1H, Nph-H), (two sets of signals).

[0214] Elemental analysis: calculated for C₂₅H₂₆ N₄ O₃ S: C, 64.91; H,5.67; N, 12.11; found: C, 65.07; H, 5.58; N, 11.93.

Example 13

[0215]N′-hydroxy-3-{[1-[(2-methyl-1-piperidinyl)carbonyl]-2-(2-naphthylsulfonyl)hydrazino]methyl}benzenecarboximidamide

[0216] The product was prepared fromN′-(3-cyanobenzyl)-N′-[(2-methyl-1-piperidinyl)carbonyl]-2-naphthalenesulfonohydrazideusing the procedure described in EXAMPLE 4.

[0217] Yield: 48%.

[0218] MP: 101-103° C.

[0219] IR (KBr): =3370, 2932, 1651 cm⁻¹.

[0220] MS (FAB): MH⁺=496.

Example 14

[0221]N′-(3-Cyanobenzyl)-6-methoxy-N′-[(2-methyl-1-piperidinyl)carbonyl]-2-naphthalenesulfonohydrazide

[0222] The product was prepared from2-(3-cyanobenzyl)-2-[(2-methyl-1-piperidinyl)carbonyl]hydraziniumchloride and 6-methoxy-2-naphthalenesulfonyl chloride using theprocedure described in EXAMPLE 3.

[0223] Yield: 26%.

[0224] MP: 169-173° C.

[0225] IR (KBr): 3172, 2934, 2229, 1671, 1623 cm⁻¹.

[0226] MS (FAB): MH⁺=493273.

[0227]¹H NMR (300 MHz, CDCl₃): 0.47-0.60 (m, 2H, Pip-H), 0.79-0.92 (m,2H, Pip-H), 1.12-1.50 (m, 6H, Pip-H in CH₃), 2.50-2.75 in 2.80-3.15 (m,1H, Pip-H), 3.55-3.76 in 3.80-3.95 (m, 1H, Pip-H), 3.95 8s, 3H, OCH₃);2.55-2.74 (m, 1H, Pip-H), 3.45-3.65 (m, 2H, Pip-H), 3.84 (s, 3H, CH₃),4.32 (br d, 2H, CH₂), 7.28-7.33 (m, 1H, Nph-H), 7.44-7.60 (m, 5H, Bn-Hand Nph-H), 7.75-7.88 (m, 3H, Nph-H), 8.34 (d, 1H, J=1.88 Hz, Nph-H).

Example 15

[0228]N′-Hydroxy-3-({2-[(6-methoxy-2-naphthyl)sulfonyl]-1-[(2-methyl-1-piperidinyl)carbonyl]hydrazino}methyl)benzenecarboximidamide

[0229] The product was prepared fromN′-(3-Cyanobenzyl)-6-methoxy-N′-[(2-methyl-1-piperidinyl)carbonyl]-2-naphthalenesulfonohydrazideusing the procedure described in EXAMPLE 4.

[0230] Yield: 45%.

[0231] MP: 172-175° C.

[0232] IR (KBr): 3368, 2937, 1647, 1160 cm⁻¹.

[0233] MS (FAB): MH⁺=526.

[0234]¹H NMR (300 MHz, CDCl₃): 0.35-0.50 (m, 2H, Pip-H), 0.79-1.00 (m,2H, Pip-H), 1.12-1.50 (m, 6H, Pip-H in CH₃), 2.40-2.75 in 2.80-3.00 (m,1H, Pip-H), 3.55-3.76 in 3.80-3.95 (m, 1H, Pip-H), 3.95 (s, 3H, OCH₃);4.35 (br d, 2H, CH₂), 5.04 (s, 2H, NH₂), 7.15-7.65 (m, 6H, Nph-H inBn-H), 7.74-7.87 (m, 3H, Nph-H), 8.34 (s, 1H, Nph-H).

[0235] Elemental analysis: calculated for C₂₆H₃₁N₅O₅S·EtOH: C, 58.83; H,6.52; N, 12.25; found: C, 59.41; H, 5.96; N, 12.09.

Example 16

[0236]N′-(3-Cyanobenzyl)-6-methoxy-N′-[(4-methyl-1-piperidinyl)carbonyl]-2-naphthalenesulfonohydrazide

[0237] The product was prepared from2-(3-cyanobenzyl)-2-[(4-methyl-1-piperidinyl)carbonyl]hydraziniumchloride using the procedure described in EXAMPLE 3.

[0238] Yield: 50%.

[0239] MP: 169-173° C.

[0240] IR (KBr): 3168, 2932, 2232, 1671 cm⁻¹.

[0241] MS (FAB): MH⁺=493.

[0242]¹H NMR (300 MHz, DMSO-d₆): 0.15-0.40 (m, 2H, Pip-H), 0.53 (d, 3H,J=6.0 Hz, CH₃), 1.25-1.39 (m, 3H, Pip-H), 2.25-2.38 (m, 1H, Pip-H),2.55-2.74 (m, 1H, Pip-H), 3.45-3.65 (m, 2H, Pip-H), 3.84 (s, 3H, CH₃),4.32 (br d, 2H, CH₂), 7.28-7.33 (m, 1H, Nph-H), 7.44-7.54 (m, 3H, Bn-Hand Nph-H), 7.60 (d, 1H, J=1.88 Hz, Bn-H), 7.60-7.76 (m, 2H, Bn-H andNph-H), 7.94 (d, 1H, J=8.66 Hz, Nph-H), 8.05 (d, 1H, J=8.66 Hz, Nph-H),8.34 (d, 1H, J=1.88 Hz, Nph-H¹), 9.46 (s, 1H, NH).

Example 17

[0243]N′-Hydroxy-3-({2-[(6-methoxy-2-naphthyl)sulfonyl]-1-[(4-methyl-1-piperidinyl)carbonyl]hydrazino}methyl)benzenecarboximidamide

[0244] The product was prepared fromN′-(3-cyanobenzyl)-6-methoxy-NV-(4-methyl-1-piperidinyl)carbonyl]-2-naphthalenesulfonohydrazideusing the procedure described in EXAMPLE 4.

[0245] Yield: 36%.

[0246] MP: 154-156° C.

[0247] IR (KBr): 3473, 3365, 2950, 1646 cm⁻¹.

[0248] MS (FAB): MH⁺=526

[0249]¹H NMR (300 MHz, CDCl₃): 0.30 (br d, 3H, Pip-H), 0.58 (d, 3H,J=6.0 Hz, CH₃), 1.26 (br d, 3H, Pip-H), 2.45 (br s, 1H, Pip-H), 2.63 (brd, 1H, Pip-H), 3.74 (br d, 2H, Pip-H), 3.97 (s, 3H, OCH₃), 4.34 (br d,2H, CH₂), 4.94 (s, 2H, NH₂) 7.17-7.35 (m, 4H, Nph-H and Bn-H), 7.49-7.55(m, 2H, Bn-H), 7.77-7.89 (m, 4H, Bn-H and Nph-H), 8.35 (m, 1H, Bn-H).8.65 (br s, 1H, NH), 9.02 (s, 1H, OH).

Example 18

[0250]Imino[3-({2-[(6-methoxy-2-naphthyl)sulfonyl]-1-[(4-methyl-1-piperidinyl)carbonyl]hydrazino}methyl)phenyl]methanaminiumChloride

[0251] The product was prepared fromN′-(3-cyanobenzyl)-6-methoxy-N′-[(4-methyl-1-piperidinyl)carbonyl]-2-naphthalenesulfonohydrazideusing the procedure described in EXAMPLE 5.

[0252] Yield: 26%.

[0253] MP: 151-154° C.

[0254] IR (KBr): 3054, 1677, 1624 cm⁻¹.

[0255] MS (FAB): MH⁺=510.

[0256]¹H NMR (300 MHz, CDCl₃): 0.28 (br d, 3H, Pip-H), 0.54 (d, 3H,J=6.0 Hz, CH₃), 1.27 (br d, 3H, Pip-H), 2.45 (br s, 1H, Pip-H), 2.64 (brs, 1H, Pip-H), 3.66 (br s, 2H, Pip-H), 3.95 (s, 3H, OCH₃), 4.32 (m, 2H,CH₂), 7.16-7.28 (m, 2H, Nph-H), 7.36-7.40 (m, 2H, Bn-H^(2,6)), 7.73-7.87(m, 4H, Bn-H⁵ and Nph-H), 7.95 (m, 1H, Bn-H⁴), 8.78 (br s, 2H, NH₂),8.16 (br s, 2H, NH₂).

Example 19

[0257]N′-(1-Azepanylcarbonyl)-N′-(3-cyanobenzyl)-2-naphthalenesulfonohydrazide

[0258] The product was prepared fromN′-(1-azepanylcarbonyl)-N′-(3-cyanobenzyl)-2-naphthalenesulfonohydrazideand naphthalene-2-sulfonyl chloride using the procedure described inEXAMPLE 3.

[0259] Yield: 43%.

[0260] MP: 128-130° C.

[0261] IR (KBr): 3190, 2942, 2223, 1674 cm⁻¹.

[0262] MS (FAB): MH⁺=463.

[0263]¹H NMR (300 MHz, DMSO-d₆): 0.63-1.40 (m, 8H, Pip-H^(3,4,5,6)),2.90-3.20 (m, 4H, Pip-H^(2,7)), 4.35 (br d, 2H, CH₂), 7.45-5.55 (m, 2H,Nph-H in Bn-H), 7.50-7-75 (m, 5H, Nph-H in Bn-H), 8.02-8.15 (m, 3H,Nph-H), 8.45 (s, 1H, Nph-H), 9.56 (s, 1H, NH).

[0264] Elemental analysis: calculated for za C₂₅H₂₆ N₄ O₃ S: C, 64.91;H, 5.67; N, 12.11; found: C, 64.65; H, 5.78; N, 12.10.

Example 20

[0265]3{[-(1-Azepanylcarbonyl)-2-(2-naphthylsulfonyl)hydrazino]methyl}-N′-hydroxybenzenecarboximidamide

[0266] The product was prepared fromN′-(1-azepanylcarbonyl)-N′-(3-cyanobenzyl)-2-naphthalenesulfonohydrazideusing the procedure described in EXAMPLE 4.

[0267] Yield: 79%.

[0268] MP: 152-155° C.

[0269] IR (KBr): 3365, 2924, 1646 cm⁻¹.

[0270] MS (FAB): MH⁺=496.

[0271]¹H NMR (300 MHz, DMSO-d₆): 0.65-1.40 (m, 8H, Pip-H^(3,4,5,6)),2.90-3.20 (m, 4H, Pip-H^(2,7)), 4.34 (br d, 2H, CH₂), 5.87 (br s, 2H,NH₂), 7.45-5.55 (m, 2H, Nph-H in Bn-H), 7.50-7-75 (m, 5H, Nph-H inBn-H), 8.02-8.15 (m, 3H, Nph-H), 8.45 (s, 1H, Nph-H), 9.35 (br d, 1H,NH), 9.67 (br s, 1H, OH).

Example 21

[0272](3{[1-(1-Azepanylcarbonyl)-2-(2-naphthylsulfonyl)hydrazino]methyl}phenyl)(imino)methanaminiumChloride

[0273] The product was prepared fromN′-(1-azepanylcarbonyl)-N′-(3-cyanobenzyl)-2-naphthalenesulfonohydrazideusing the procedure described in EXAMPLE 5.

[0274] Yield: 36%.

[0275] MP: 151-154° C.

[0276] IR (KBr): 3374, 3054, 2926, 1654, 1420, 1160 cm¹.

[0277] MS (FAB): MH⁺=480.

[0278]¹H NMR (300 DMSO-d₆): 0.60-1.45 (m, 8H, Pip-H^(3,4,5,6)),2.90-3.20 (m, 4H, Pip-H^(2,7)), 4.40 (br d, 2H, CH₂), 7.44 (d, J=8,3 Hz,2H, Bn-H 2,6), 7.62-7.80 (m, 5H, Bn-H^(3,5) in Nph-H), 8.02-8.18 (m, 3H,Nph-H), 8.45 (s, 1H, Nph-H), 9.09 (s, 2H, NH₂), 9.35 (s, 1H, NH)

Example 22

[0279]N′-(1-Azepanylcarbonyl)-N′-(3-cyanobenzyl)-6-methoxy-2-naphthalenesulfonohydrazide

[0280] The product was prepared from2-(3-cyanobenzyl)-2-[(4-methyl-1-piperidinyl)carbonyl]hydraziniumchloride using the procedure described in EXAMPLE 3.

[0281] Yield: 15%.

[0282] MP: 128-130° C.

[0283] IR (KBr): 3169, 2939, 2229, 1670 cm⁻¹.

[0284] MS (FAB): MH⁺=493.

[0285]¹H NMR (300 MHz, DMSO-d₆): 0.65-1.50 (m, 8H, Pip-H^(3,4,5,6)),2.85-3.20 (m, 4H, Pip-H^(2,7)), 3.91 (s, 3H, OCH₃) 4.35 (br d, 2H, CH₂),7.28-7.32 (m, 1H, Bn-H), 7.42-7.52 (m, 3H, Bn-H in Nph-H), 7.60 (br s,1H, Nph-H), 7.66-7.71 (m, 2H, Nph-H), 7.92 (d, 1H, J=8.7, Nph-H), 8.04(d, 1H, J=8.7, Nph-H), 8.33 (d, 1H, J=1.5, Nph-H), 9.44 (s, 111, NH).

[0286] Elemental analysis: calculated for C₂₆H₂₈ N₄ O₄ S: C, 63.14; H,6.11; N, 11.33; found: C, 63.26; H, 5.87; N, 11.06.

Example 23

[0287]3-({1-(1-Azepanylcarbonyl)-2-[(6-methoxy-2-naphthyl)sulfonyl]hydrazino}methyl)-N′-hydroxybenzenecarboximidamide

[0288] The product was prepared fromIV-(1-azepanylcarbonyl)-N′-(3-cyanobenzyl)-6-methoxy-2-naphthalenesulfonohydrazideusing the procedure described in EXAMPLE 4.

[0289] Yield: 24%.

[0290] MP: 173-175° C.

[0291] IR (KBr): 3459, 3368, 2932, 1648, cm⁻¹.

[0292] MS (FAB): MH⁺=526.

[0293]¹H NMR (300 MHz, CDCl₃): 0.65-1.50 (m, 8H, Pip-H^(3,4,5,6)),2.85-3.20 (m, 4H, Pip-H^(2,7)), 3.97 (s, 3H, OCH₃) 4.35 (s, 2H, CH₂),4.95, (s, 1H, NH) 7.28-7.32 (m, 1H, Bn-H), 7.42-7.52 (m, 3H, Bn-H inNph-H), 7.60 (br s, 1H, Nph-H), 7.66-7.71 (m, 2H, Nph-H), 7.92 (d, 1H,J=8.7 Hz, Nph-H), 8.04 (d, 1H, J=8.7 Hz, Nph-H), 8.33 (d, 1H, J=1.5 Hz,Nph-H), 8.10 (br s, 1H, NH), 8.35 (s, 1H, OH).

[0294] Elemental analysis: calculated for C₂₆H₃₁N₅ O₅ S: C, 59.18; H,6.30; N, 13.27; found: C, 59.68; H, 6.16; N, 12.88.

Example 24

[0295][3-({1-(1-Azepanylcarbonyl)-2-[(6-methoxy-2-naphthyl)sulfonyl]hydrazino}methyl)phenyl](imino)methanaminiumChloride

[0296] The product was prepared fromN′-(1-azepanylcarbonyl)-N′-(3-cyanobenzyl)-6-methoxy-2-naphthalenesulfonohydrazideusing the procedure described in EXAMPLE 5.

[0297] Yield: 21%.

[0298] MP: 151-154° C.

[0299] IR (KBr): 3057, 2929, 1677, 1623, 1474, 1157 cm⁻¹.

[0300] MS (FAB): MH⁺=510.

[0301]¹H NMR (300 MHz CDCl₃): 0.65-1.40 (m, 8H, Pip-H^(3,4,5,6)),2.95-3.20 (m, 4H, Pip-H^(2,7)), 3.97 (s, 3H, OCH₃) 4.35 (br s, 2H, CH₂),7.44 (d, J=8,3 Hz, 2H, Bn-H^(2,6)), 7.62-7.80 (m, 5H, Bn-H^(3,5) inNph-H), 8.02-8.18 (m, 3H, Nph-H), 8.45 (s, 1H, Nph-H), 9.03 (s, 2H,NH₂), 9.35 (s, 2H, NH₂).

[0302] Results of Biological in Vitro Testing for Selected Compounds Kitrypsin/ Ki thrombin Ki trypsin Ki aPTT PT TT (μM) (μM) thrombin (μM)(μM) (μM)

0.469 >68.3 146 — — —

0.004 0.112 28 — — —

0.011 — — 2.1 2.7 2.0

0.594 32.236 54 — — —

0.005 0.143 29 — — —

0.61 17.945 29 — — —

0.009 0.053 5.84 — — —

Example 27

[0303] Results of Biological in Vivo Testing for Selected CompoundsStasis-induced thrombosis Bleeding (ED₅₀ ± SD − mg/kg) (fold increase at1 mg/kg)

290 ± 11 3.3

 70 ± 5 9.4

1. The compounds of the general formula I

wherein R¹ represents a residue of the formula

 wherein R⁴=H, alkyl (C₁-C₃), OH, O-alkyl (C₁-C₃), NH₂; R² represents aresidue of the formula

 wherein R⁵H, alkyl (C₁-C₃), CF₃, COOR⁹, R⁶=H, alkyl (C₁-C₃), CF₃,COOR⁹, R⁷=alkyl (C₁-C₃), cycloalkyl (C₃-C₆), R⁸=alkyl (C₁-C₃),cycloalkyl (C₃-C₆), R⁹=H, alkyl (C₁-C₃), R¹⁰=H, alkyl (C₁-C₃), benzyl,R¹¹=H, SO₂Me, CO₂Me R³ represents a residue of the formula

 wherein R¹²=H, alkyl (C₁-C₃), acetyl  and pharmaceutically acceptablesalts thereof.
 2. A compound as claimed in claim 1, of formula I whereinR² represents a group selected from:


3. A compound as claimed in preceding claim wherein R³ represents agroup selected from:


4. A compound as claimed in claim 1 wherein said compound is selectedfrom the group consisting of:N′-hydroxy-3-{[1-[(2-methyl-1-piperidinyl)carbonyl]-2-(2-naphthylsulfonyl)hydrazino]methyl}benzenecarboximidamideN′-Hydroxy-3-({2-[(6-methoxy-2-naphthyl)sulfonyl]-1-[(2-methyl-1-piperidinyl)carbonyl]hydrazino}methyl)benzenecarboximidamideImino[3-({2-[(6-methoxy-2-naphthyl)sulfonyl]-1-[(2-methyl-1-piperidinyl)carbonyl]hydrazino}methyl)phenyl]methanaminium chlorideN′-Hydroxy-3-{[1-[(4-methyl-1-piperidinyl)carbonyl]-2-(2-naphthylsulfonyl)hydrazino]methyl}benzenecarboximidamideImino(3-{[1-[(4-methyl-1-piperidinyl)carbonyl]-2-(2-naphthylsulfonyl)hydrazino]methyl}phenyl)methanaminium chlorideN′-Hydroxy-3-({2-[(6-methoxy-2-naphthyl)sulfonyl]-1-[(4-methyl-1-piperidinyl)carbonyl]hydrazino}methyl)benzenecarboximidamideImino[3-({2-[(6-methoxy-2-naphthyl)sulfonyl]-1-[(4-methyl-1-piperidinyl)carbonyl]hydrazino}methyl)phenyl]methanaminium chloride3-{[1-(1-Azepanylcarbonyl)-2-(2-naphthylsulfonyl)hydrazino]methyl}-N′-hydroxybenzenecarboximidamide(3-{[1-(1-Azepanylcarbonyl)-2-(2-naphthylsulfonyl)hydrazino]methyl}phenyl)(imino)methanaminium chloride3-({1-(1-Azepanylcarbonyl)-2-[(6-methoxy-2-naphthyl)sulfonyl]hydrazino}methyl)-N′-hydroxybenzenecarboximidamide[3-({1-(1-Azepanylcarbonyl)-2-[(6-methoxy-2-naphthyl)sulfonyl]hydrazino}methyl)phenyl](imino)methanaminium chloride.
 5. Compounds of the formula Iaccording to claim 1 for use in therapy.
 6. A process for preparingcompounds of the formula I according to claim 1, characterized in that3-cyanobenzaldehyde of the formula (II)

is converted with BOC-carbazate of the formula (III)

to the compound of the formula (IV),

which is subjected to reduction by catalytic hydrogenation or withNaCNBH₃ is converted to the compound (V),

which reacts with triphosgene and amine of the formula (VI),

wherein R⁵ and R⁶ have the same meanings as in the formula (I), or withtriphosgene and amine of the formula (VII or VII),

or with triphosgene and amine of the formula (IX),

wherein R⁷ and R⁸ have the same meanings as in the formula (I), or withtriphosgene and amine of the formula (X),

wherein R¹⁰ has the same meaning as in the formula (I), in “one pot” upto the compound (XI),

wherein R² has the same meaning as in formula (I); in which theprotecting BOC group is removed with HCl (g) in HOAc at room temperatureto obtain the compound (XII),

wherein R has the same meaning as in the formula (I), which reacts witharomatic sulfonylchloride up to the compound (XIII)

wherein R² and R³ have the same meanings as in the formula (I); whichare then converted to the compound of the formula (I).


7. The use of compounds of the formula I of claim 1, in the manufactureof medicaments which inhibit thrombin and formation of fibrin andthrombin.
 8. Pharmaceutical compositions comprising a therapeuticallyeffective amount of the compound of the formula I of claim 1 andpharmaceutically acceptable auxiliary substances.
 9. Pharmaceuticalcompositions according to claim 8, for inhibiting thrombin in man andother mammals.
 10. Pharmaceutical compositions according to claim 8, forinhibiting formation of fibrin and thrombin in the blood of man andother mammals.
 11. A method for inhibiting thrombin in the blood of manand other mammals comprising administering a compound of the formula Iof claim 1
 12. A method for inhibiting formation of fibrin and thrombinin man and other mammals comprising administering a compound of theformula I of claim 1.